4-isopropyltoluene-based bactrocera dorsalis repellent and use thereof

Abstract

The present application belongs to the field of bio-technology of forestry and agricultural insects, and provides a kind of 4-isopropyl toluene-based Bactrocera dorsalis repellent and its application.The effective component of the repellent is 1%-16% plant-derived volatile, selected from 4-isopropyl toluene, trans-caryophyllene, carvone, menthone, eucalyptol, linalool and other plant-derived volatiles, preferably 16% 4-isopropyl toluene, or a 3:1 compounded system of 4-isopropyl toluene and eucalyptol.16% 4-isopropyl toluene has the best repellent effect, can simultaneously repel male and female adults of Bactrocera dorsalis, inhibit oviposition of female adults, and has a broad-spectrum and stable repellent effect on multiple host fruits such as oranges, mangoes and guavas;the olfactory responses of male and female adults are highly consistent, and the olfactory repellent effect is significantly correlated with the oviposition inhibition effect.The present application confirms the potential of 4-isopropyl toluene as a highly effective repellent for Bactrocera dorsalis, and provides a new type of plant-derived preparation for the non-polluting control of Bactrocera dorsalis.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and forestry insect biotechnology, and relates to a repellent based on plant-derived volatiles, specifically a repellent for the oriental fruit fly based on 4-isopropyltoluene and its application. Background Technology

[0002] The oriental fruit fly (Bactrocera dorsalis (Hendel)) is a major devastating pest of tropical and subtropical fruit trees such as citrus, mango, and guava in southern my country. Adult females lay eggs inside ripe fruit using their ovipositors, while the larvae bore into the fruit flesh, causing rot and fruit drop, severely reducing yield and commercial value. Currently, the control of the oriental fruit fly mainly relies on frequent spraying of chemical insecticides. However, the long-term and excessive use of chemical pesticides easily leads to three major problems: first, the oriental fruit fly population rapidly evolves resistance to pesticides, and the efficacy of conventional pesticides declines year by year; second, pesticide residues exceed standards, threatening the safety of fruit consumption and import / export trade; and third, broad-spectrum chemical pesticides kill natural enemies such as parasitic wasps and predatory insects in orchards, disrupting the ecological balance of the orchard.

[0003] Plant-derived volatiles, derived from natural plant essential oils, possess advantages such as low toxicity, easy degradation, good environmental compatibility, and low likelihood of developing resistance, making them a core research direction for green pest control formulations. Existing research confirms that various terpenoid volatiles can regulate the olfactory and oviposition behaviors of fruit flies, but current technologies have significant drawbacks: First, most volatiles only repel female or male fruit flies individually, failing to simultaneously inhibit the activity of adult females and males, resulting in limited population control effects; second, many terpenoids exhibit a dual concentration effect, attracting fruit flies at low concentrations but only possessing weak repellent activity at high concentrations, lacking stable, broad-spectrum repellent components; third, existing repellents only achieve olfactory repellency, failing to significantly inhibit female oviposition and thus unable to reduce the initial population size; fourth, most repellent components are only effective against a single host fruit tree, resulting in a narrow range of applications and poor formulation versatility.

[0004] Current literature reports that monoterpenoids such as 4-isopropyltoluene and eucalyptol have repellent activity against stored pests. However, there has been no systematic comparison of the differences in olfactory behavior of male and female Bactrocera dorsalis var. citrinum volatiles of nine typical plants. The optimal concentration of each component has not been determined, nor has the broad-spectrum oviposition inhibition of single-component and binary compound systems on multiple host fruits been verified. There is a lack of plant-derived repellents specifically for Bactrocera dorsalis var. citrinum that have both sexual repellency and broad-spectrum oviposition inhibition effects. Summary of the Invention

[0005] The purpose of this invention is to provide a repellent for the oriental fruit fly based on 4-isopropyltoluene and its application. By systematically screening plant-derived volatiles and determining the optimal application concentration and compound ratio, it can simultaneously repel both male and female oriental fruit flies and inhibit oviposition by female flies. It has a stable repellent effect on multiple host fruits such as oranges, mangoes, and guavas. It is green, safe, and environmentally friendly, and is suitable for green pest control in orchards.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a repellent for the oriental fruit fly based on plant-derived volatiles, wherein the effective component of the oriental fruit fly repellent is a plant-derived volatile, the volume fraction of which is 1%-32%, and the plant-derived volatile is at least one selected from 4-isopropyltoluene, trans-caryophyllene, carvone, menthone, eucalyptol, and linalool.

[0007] Preferably, the plant-derived volatiles are at least one of 4-isopropyltoluene and eucalyptol.

[0008] Preferably, the plant-derived volatile compound is a single 4-isopropyltoluene, and the 4-isopropyltoluene has a volume fraction of 16%.

[0009] Preferably, the plant-derived volatile compound is a single eucalyptol, and the volume fraction of the eucalyptol is 16%.

[0010] Preferably, the plant-derived volatiles are a mixture of 4-isopropyltoluene and eucalyptol, with a volume ratio of (1-3):(1-3).

[0011] Preferably, the volume ratio of 4-isopropyltoluene to eucalyptol is 3:1.

[0012] Preferably, the oriental fruit fly repellent simultaneously repels both male and female oriental fruit fly adults and inhibits oviposition by females.

[0013] Preferably, the oriental fruit fly repellent also includes pesticide-acceptable excipients.

[0014] Preferably, the excipients are selected from at least one of solvents, solubilizers, emulsifiers, stabilizers, osmotic pressure regulators, pH buffers, surfactants, diluents, and suspending agents.

[0015] Preferably, the oriental fruit fly repellent is any one of the following: aqueous solution, emulsifiable concentrate, microcapsule, dispersible oil suspension, granules, or ointment.

[0016] The beneficial effects of this invention are:

[0017] 1. The active ingredients have a broad spectrum of compatibility with both male and female fruit flies, exhibiting high repellency consistency. Spearman correlation analysis confirmed a highly significant positive correlation (r0.05) between the olfactory responses of male and female adult fruit flies to the volatiles of this invention. s=0.9802, P<0.0001), the repellent can act on both male and female individuals in the population simultaneously, avoiding the control loopholes caused by single-sex repellency and improving the overall population suppression effect.

[0018] 2. The optimal concentration window for action is clearly defined, and application costs are controllable. The repellent activity of 4-isopropyltoluene and eucalyptol is significantly concentration-dependent, with 16% by volume being the optimal application concentration. Increasing the concentration to 32% and 64% does not improve the repellent effect, so high concentrations are not required in the field, reducing the cost of raw materials.

[0019] 3. It possesses both olfactory repellency and oviposition inhibition functions, controlling the problem at its source. The olfactory behavior index of female insects is significantly positively correlated with the oviposition index (r...). s =0.8645, P<0.0001), the olfactory repulsion level can directly predict the oviposition inhibition effect; 16% 4-isopropyltoluene oviposition index OI=-0.59 can reduce the number of female eggs laid, thus suppressing the fruit fly population from the reproductive end.

[0020] 4. Wide-spectrum applicability to multiple hosts and strong formulation versatility. 16% 4-isopropyltoluene exhibits oviposition-repelling effects on three typical host fruits: orange, mango, and guava. One formulation can be adapted to multiple types of tropical fruit trees, simplifying the variety of pesticides available in orchards.

[0021] 5. The repellent activity of the core component is superior to that of the binary compound system, and the formulation is simple and stable. The optimal compound ratio is 3:1 of 4-isopropyltoluene and eucalyptol, but the repellent effect of all compound schemes is weaker than that of 16% 4-isopropyltoluene alone. 4-isopropyltoluene is the core functional substance, and the preparation process of the single-agent formulation is simple, with no risk of component antagonism.

[0022] 6. Natural plant-derived components, green, safe, and residue-free. The active ingredients are natural terpenoid volatiles from plant essential oils, which are easily degraded in the natural environment, will not cause pesticide residues on fruits, will not kill natural enemy insects in orchards, are compatible with green and organic orchard production standards, and avoid problems such as chemical pesticide resistance and trade barriers. Attached Figure Description

[0023] Figure 1 This is the experimental design diagram of the Y-tube in this invention;

[0024] Figure 2 This is the design diagram of the oviposition selection experiment in this invention;

[0025] Figure 3 This invention describes the olfactory behavioral response of adult oriental fruit flies to plant-derived volatiles (Figure A shows the olfactory behavioral response of female flies; Figure B shows the olfactory behavioral response of male flies).

[0026] Figure 4This invention describes the olfactory behavioral responses of the oriental fruit fly to different concentrations of 4-isopropyltoluene (Figure A shows the olfactory behavioral responses of the female fly; Figure B shows the olfactory behavioral responses of the male fly).

[0027] Figure 5 This invention describes the olfactory behavioral responses of the oriental fruit fly to different concentrations of eucalyptol (Figure A shows the olfactory behavioral responses of the female fly; Figure B shows the olfactory behavioral responses of the male fly).

[0028] Figure 6 This invention relates to the correlation analysis of the behavioral indices of male and female oriental fruit flies (each point in the figure represents a combination of a compound and its concentration; color indicates compound AI, and symbols indicate concentrations of 1%, 4%, and 16%; solid lines show the fitted relationship).

[0029] Figure 7 This invention presents a correlation analysis of the behavioral index and oviposition index of female oriental fruit fly (each point in the figure represents a combination of compound and concentration; colors represent compounds A, E, F, G, H, and I, and symbols represent concentrations of 1%, 4%, and 16%; solid lines show the fitted relationship).

[0030] Figure 8 This invention describes the oviposition selection response of the oriental fruit fly to a mixture of 4-isopropyltoluene (A3) and eucalyptol (H3) (in the figure, T1-T5 represent A3:H3 ratios of 1:3, 1:2, 1:1, 2:1, and 3:1, respectively; Figure A shows the average oviposition in the treatment and control groups, and Figure B shows the oviposition index for each mixture; data are expressed as average oviposition ± standard error, and different lowercase letters indicate significant differences between different treatments, p < 0.05).

[0031] Figure 9 This invention describes the oviposition repellency activity of 4-isopropyltoluene, eucalyptol, and their mixtures against the oriental fruit fly (A3 and H3 in the figure represent 16% 4-isopropyltoluene and 16% eucalyptol, respectively, and T5 represents a 3:1 binary mixture; data are expressed as mean oviposition ± standard error; * indicates significant difference between treatments (P<0.05), ** indicates extremely significant difference between treatments (P<0.01), and ns indicates no significant difference).

[0032] Figure 10This invention describes the inhibitory effect of 4-isopropyltoluene on oviposition of *Bactrocera dorsalis* on different host fruits (Figure A shows the inhibitory effect of 4-isopropyltoluene on oviposition of *Bactrocera dorsalis* on different host fruits; data are expressed as mean oviposition ± standard error, *** indicates highly significant difference between the treatment group and the control group (P<0.001); Figure B shows the oviposition selection test using orange pulp as the oviposition substrate; Figure C shows the oviposition selection test using mango pulp as the oviposition substrate; Figure D shows the oviposition selection test using guava pulp as the oviposition substrate; where CK represents the control group, A3 represents the treatment group, and the white strips are *Bactrocera dorsalis* eggs; each treatment was repeated 5 times). Detailed Implementation

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Example 1: Screening of 4-isopropyltoluene, a plant-derived repellent for the oriental fruit fly.

[0037] 1. Materials and Methods

[0038] 1.1 Test insect source

[0039] The tested *Bactrocera dorsalis* flies had been artificially reared in the laboratory for over 20 generations. Rearing conditions were: temperature 27±1℃, relative humidity 65±5%, and photoperiod L:D = 14 h:10 h. Larvae were fed bananas. When the larvae reached the third instar, they were transferred to plastic boxes (20 cm × 12 cm × 8 cm) filled with damp sand to pupate. Approximately 7 days later, before emergence, the pupae were collected into petri dishes (9 cm × 9 cm) and placed in rearing cages (35 cm × 35 cm × 35 cm) to facilitate adult emergence. They were fed an artificial diet consisting of a 1:3 mixture of yeast powder and sucrose, and water was provided. Adults appearing within 24 hours were considered to be 0 days old. After emergence, males and females were separated and reared individually. 12-day-old males and females were placed in the same cage for mating. Experiments were conducted using 15-day-old mated males and females.

[0040] 1.2 Test Reagents

[0041] 4-Isopropyltoluene, 3-carene, α-pinene, β-pinene, trans-caryophyllene, carvone, menthone, eucalyptol, and linalool were selected as candidate repellents. These compounds are mostly monoterpenes or sesquiterpenes, important components of plant essential oils, and play a crucial role in plant-insect interactions. α-pinene exhibits significant repellent and oviposition-inhibiting effects against the oriental fruit fly (Bactrocera dorsalis), while β-pinene and linalool also show significant repellent activity; sesquiterpenes such as trans-caryophyllene are involved in the olfactory recognition process of fruit flies. Furthermore, 4-isopropyltoluene, carvone, menthone, and eucalyptol showed good repellent or toxic activity against various pests. All volatile compounds from the tested plants were commercially available. In the experiments, all plant volatile compounds were prepared into 1%, 4%, and 16% (v / v) solutions using n-hexane as the solvent. In the following groups, the letters AI represent 4-isopropyltoluene, 3-carene, α-pinene, β-pinene, trans-caryophyllene, carvone, menthone, eucalyptol, and linalool, respectively; the numbers 1-3 represent concentrations of 1%, 4%, and 16%, respectively. For example, A1 represents 1% 4-isopropyltoluene, A2 represents 4% 4-isopropyltoluene, and A3 represents 16% 4-isopropyltoluene.

[0042] 1.3 Olfactory Behavioral Response of the Oriental Fruit Fly to Plant Volatile Compounds

[0043] The behavioral responses of adult female and male oriental fruit flies to plant volatiles were measured using a Y-shaped olfactory apparatus. The Y-shaped tube, made of colorless, transparent glass, consisted of one adaptation arm and two test arms, each 15 cm long, angled at 75° with a 2.5 cm inner diameter. Each test arm was connected to a glass odorant bottle (3.5 cm in diameter × 6.5 cm in length). The apparatus was powered by an air pump. Figure 1 Following the airflow direction, the components of the device are, in sequence, an air pump, a flow meter, an adsorption activated carbon drying tower, sterile water, a flavor source bottle, and a Y-shaped glass tube. All components are connected by transparent plastic tubing. The airflow velocity within the device is controlled at 175 mL / min. -1 .

[0044] 100 μL of the test solution was dropped onto filter paper (2 cm × 2 cm) and placed in a flavor source bottle as the treatment group. Another flavor source bottle contained filter paper with 100 μL of n-hexane added as the control group. The test insects were starved for 3 h before the experiment. One adult oriental fruit fly was placed in each experiment and observed at the opening of the Y-shaped adaptation arm. When the test insect passed halfway through one arm of the Y-shaped tube and remained there for more than 30 seconds, it was considered to have made a selection; if the test insect did not make a selection within 10 minutes of entering the adaptation arm, it was considered not to have selected. To minimize positional bias, the positions of the flavor source bottle and test arm were swapped after every 5 oriental fruit flies were tested. After each experiment, the Y-shaped tube apparatus was cleaned with acetone and dried in an oven. Each test insect was tested only once, and each group of experiments was repeated 60 times. All experiments were conducted between 14:00 and 18:00, with an indoor temperature of 27±1℃. Incandescent lamps (15 W) were used to uniformly illuminate the instruments during the experiments to eliminate the influence of phototaxis in the oriental fruit fly.

[0045] 1.4 Selective oviposition response of the oriental fruit fly to plant volatiles

[0046] This experiment was conducted in an artificial climate chamber, and the specific procedures are as follows ( Figure 2 Fresh banana pulp was used as the oviposition substrate. 5 g of banana pulp was weighed and placed in a transparent plastic cup (3.0 cm × 3.0 cm). 100 μL of the test solution was added to the treatment cup and thoroughly mixed with the banana pulp; 100 μL of n-hexane was added to the control cup in the same manner. Several small holes were pierced on the side wall of each plastic cup to allow female oriental fruit flies to insert into their ovipositors to lay eggs. One treatment cup and one control cup were set up for each replicate, placed diagonally opposite each other in a rearing cage (30 cm × 30 cm × 30 cm). Ten mated female adults were placed in each cage, allowing them to freely choose where to lay eggs. After 24 h, the number of eggs laid in the treatment and control cups was counted. Each treatment was repeated 5 times.

[0047] 1.5 Oviposition Selection Response of the Oriental Fruit Fly to Binary Mixtures with Different Proportions

[0048] Based on the results of previous repellency behavior and oviposition repellency tests, 16% 4-isopropyltoluene (A3) and 16% eucalyptol (H3), which showed good repellency effects, were selected for mixture testing. Five binary mixtures of A3 and H3 were prepared by volume ratio: T1 (A3:H3=1:3), T2 (1:2), T3 (1:1), T4 (2:1), and T5 (3:1). The oviposition repellency activity of the five mixtures was evaluated using the above-mentioned dual-selection oviposition test method to screen the optimal ratio.

[0049] To further compare the repellency activities of key candidates, a dual-selection oviposition test was conducted on A3, H3 and their optimal mixture (T5) to compare their oviposition repellency effects.

[0050] 1.6 The repellent effect of plant volatiles on oviposition of the oriental fruit fly on different host fruits

[0051] To verify whether the repellent effect of the optimal repellent compound is consistent across different host fruits, fresh orange, mango, and guava pulps (5 g / cup) were prepared, using the same treatment method as described in 2.1.4. 100 μL of A3 was added to each pulp cup as a treatment, and 100 μL of n-hexane was added to the control cup. The determination was performed according to the double-selection oviposition test method described above.

[0052] 1.7 Data Statistical Analysis

[0053] The behavioral response of adult female and male oriental fruit flies to plant volatiles was assessed using the Preference Index (PI). The PI calculation formula is as follows:

[0054]

[0055] In the formula, T is the number of insects selected for the treatment arm; C is the number of insects selected for the control arm; PI values ​​from -1.00 to -0.10 are considered repulsive; PI values ​​from -0.10 to +0.10 are considered neutral; and PI values ​​from +0.10 to +1.00 are considered attractive.

[0056] The spawning selection response is represented by the Oviposition Index (OI), which is calculated using the following formula:

[0057]

[0058] In the formula, T represents the total number of eggs laid in the treatment cup; C represents the total number of eggs laid in the control cup; a positive OI value indicates a preference for egg laying in the treatment; a negative value indicates an egg-laying repellent effect of the treatment; and 0 indicates no selectivity.

[0059] For olfactory behavioral response data, the chi-square test was used to analyze the selection differences between the treatment group and the control group, with a significance level set at α=0.05. Only individuals of the oriental fruit fly that made a selection were included in the analysis; individuals that did not make a selection were not included in the statistical analysis. For the oviposition selection experiment, a paired-samples t-test was used to compare the difference in oviposition between the treatment group and the control group, with a significance level set at α=0.05. For oviposition experiments involving multiple treatment groups, one-way ANOVA was used to compare the average oviposition of each treatment group. After the ANOVA showed significance, Tukey's multiple comparison method was used for post-hoc testing, with a significance level set at α=0.05. To explore the association between olfactory behavioral responses and oviposition behavioral responses, Spearman correlation analysis was used to analyze the correlation between female and male behavioral indices, and between female and oviposition indices.

[0060] 2. Results Analysis

[0061] 2.1 Olfactory behavioral response of the oriental fruit fly to plant volatiles

[0062] The olfactory behavioral responses of adult citrus fruit flies to volatiles from nine tested plants showed significant differences. Among females ( Figure 3 In Group A, treatments with 16% concentrations of 3-carene, α-pinene, and β-pinene significantly increased the female insects' selection rate for the treated arms, with preference indices of 0.30, 0.27, and 0.27, respectively. At the same concentrations, 16% concentrations of 4-isopropyltoluene, carvone, menthone, and eucalyptol significantly reduced the female insects' selection for the treated arms, with 4-isopropyltoluene showing the strongest repellent effect (PI = -0.40). Furthermore, trans-caryophyllene and linalool showed significant attractant effects on female insects at low concentrations of 1% (PIs of 0.33 and 0.27, respectively), but their attractant effect weakened or even transformed into a neutral or repellent effect with increasing concentration.

[0063] In male insects ( Figure 3 In the middle (B) concentration, 16% of 3-carene, α-pinene, and β-pinene also showed significant attraction (PI = 0.30, 0.27, and 0.27, respectively), while 16% of 4-isopropyltoluene significantly reduced the male insects' selection for the treated arm (PI = -0.27). Similar to female insects, trans-caryophyllene and linalool had an attraction effect on male insects at 1% concentration, but this effect weakened at higher concentrations.

[0064] Further dose-response tests were conducted on 4-isopropyltoluene and eucalyptol. Figure 4 , Figure 5 The results showed that the repellent effect of both was strongest at a concentration of 16%, and the repellent effect did not increase further when the concentration was increased to 32% and 64%. 16% 4-isopropyltoluene showed significant repellent effects on both female and male insects (PIs of -0.37 and -0.30, respectively), while the 32% and 64% concentrations showed no significant repellent effect. Eucalyptol showed a similar trend: a 16% concentration showed significant repellent effects on both male and female insects (PI of -0.27 for both), but the 32% and 64% concentrations did not show significant repellent activity.

[0065] 2.2 Correlation analysis of olfactory behavioral responses of male and female oriental fruit flies to plant volatiles

[0066] To investigate whether there are differences in the olfactory behavioral responses of male and female oriental fruit fly (Bactrocera dorsalis) and whether this has a potential impact on repellent selection, correlation analysis was performed on the behavioral indices (PIs) of male and female oriental fruit fly volatiles at three concentrations of nine plant volatiles. The results showed that ( Figure 6There is a highly significant positive correlation between the two (r) s =0.9802, P<0.0001), indicating a high degree of consistency in the olfactory behavioral responses of male and female adults to the nine tested volatiles. Based on this result, it is speculated that male and female oriental fruit fly larvae may share similar receptors and neural coding mechanisms during olfactory recognition, providing a theoretical basis for subsequent screening of repellents with broad-spectrum activity in both sexes, while also reducing the influence of sex differences.

[0067] 2.3 Effects of plant volatiles on oviposition selection in the oriental fruit fly

[0068] Six volatiles with repellency potential were screened from the olfactory response behavior of the oriental fruit fly to plant volatiles: (A) 4-isopropyltoluene, (E) trans-caryophyllene, (F) carvone, (G) menthone, (H) eucalyptol, and (I) linalool. Further oviposition selection experiments were conducted on these six volatiles, and the results are shown in Table 1. At the three tested concentrations, only the treatments with 16% 4-isopropyltoluene (A3) and 16% eucalyptol (H3) showed significantly lower oviposition compared to the control group. A3 exhibited the most significant oviposition repellency effect, with 47.80±6.87 eggs laid in the treatment group compared to 188.00±11.40 eggs in the control group, and an oviposition index (OI) of -0.59±0.07 (P=0.002). The H3 treatment group also showed a significant egg-laying repellency effect, with 99.80±8.77 eggs laid in the treatment group and 167.40±22.09 eggs laid in the control group. OI=-0.28±0.06 (P=0.021).

[0069] Carvone and menthone showed negative oviposition indices at all tested concentrations, indicating a certain oviposition repellency trend, but no significant difference was found between the treatment groups and the control group (P>0.05). In contrast, the oviposition rates in the 1% trans-caryophyllene (E1) and 1% linalool (I1) treatment groups were significantly higher than those in the control group, with oviposition indices of 0.26±0.06 (P=0.006) and 0.18±0.05 (P=0.018), respectively, demonstrating significant oviposition attraction.

[0070] Table 1. Oviposition selection experiment of female oriental fruit fly (Bactrocera dorsalis) on plant-derived volatiles.

[0071]

[0072] Note: A, E, F, G, H, and I represent 4-isopropyltoluene, trans-caryophyllene, carvone, menthone, eucalyptol, and linalool, respectively; 1, 2, and 3 represent 1%, 4%, and 16% concentrations, respectively; values ​​are expressed as mean egg production ± standard error; P-values ​​represent paired t-tests between the treatment and control groups; * indicates a significant difference between the treatment and control groups (P < 0.05); ** indicates an extremely significant difference (P < 0.01).

[0073] 2.4 Correlation analysis of the oviposition and olfactory repellency effects of plant volatiles on the oriental fruit fly.

[0074] To investigate the relationship between olfactory response and oviposition selection in the oriental fruit fly (Bactrocera dorsalis) and to clarify whether rapid olfactory behavior measurement can predict oviposition repellency, correlation analysis was performed on the female behavioral index (PI) and oviposition index (OI) of six plant-derived volatiles at different concentrations. Figure 7 There is a highly significant positive correlation between the two (r). s =0.8645, P<0.0001). The experimental results show that volatiles that elicit strong repellent behavior in females during olfactory behavior also exhibit a strong oviposition repellency effect in oviposition selection experiments, indicating a consistency between olfactory and oviposition behavioral responses. This finding provides an important basis for simplifying the repellent screening process, namely, that the oviposition repellency potential of volatiles can be preliminarily assessed through rapid olfactory behavior measurements. It also suggests that there may be a common neural regulatory pathway between olfactory recognition and oviposition decision-making in the oriental fruit fly during the perception of repellent substances.

[0075] 2.5 The oviposition-repelling effect of binary mixtures with different ratios on the oriental fruit fly (Bactrocera dorsalis)

[0076] Both 16% 4-isopropyltoluene and 16% eucalyptol exhibited significant behavioral and oviposition-repellent effects against the oriental fruit fly. Therefore, five binary mixtures were prepared by varying proportions of 4-isopropyltoluene (A3) and eucalyptol (H3) for oviposition selection experiments to screen for the optimal ratio. The results showed that ( Figure 8 Among the five mixtures, the T5 treatment group (A3:H3=3:1) had the lowest egg production, with 72.00±9.26 eggs in the treatment group and 181.00±12.24 eggs in the control group, and the lowest egg production index (-0.43±0.06). Multiple comparison analysis showed that the egg-laying repellency effect of T5 was significantly higher than that of the T1-T4 treatment groups (F=9.429; df=4; P=0.0002), while there were no significant differences among the four T1-T4 groups.

[0077] The oviposition repellency activities of A3, H3, and their optimal mixture T5 were further compared. The results showed that ( Figure 9The oviposition repellency effect of A3 was significantly stronger than that of H3 and T5, while there was no significant difference between T5 and H3. The experimental results indicate that the oviposition repellency effect of the binary mixture of A3 and H3 is lower than that of A3, and A3 is the key plant volatile that repels the oriental fruit fly.

[0078] 2.6 The repellent effect of plant volatiles on oviposition of the oriental fruit fly on different host fruits

[0079] To verify whether the oviposition repellency effect of 16% 4-isopropyltoluene (A3) was consistent on different host fruits, oviposition selection experiments were conducted using orange, mango, and guava pulp as oviposition substrates. The results showed that ( Figure 10 (A), on the fruits of the three tested host plants ( Figure 10 China B- Figure 10 In the D and A3 treatment groups, the number of eggs laid was significantly lower than that in the control group (P<0.001), indicating that A3 has a broad-spectrum and consistent egg-repelling effect on different host fruits.

[0080] In summary, this invention, relying on a Y-type olfactometer behavior measurement and oviposition selectivity test system, evaluated the olfactory tropism and oviposition inhibition activities of nine plant-derived volatiles on adult oriental fruit flies. It confirmed that different volatiles exhibited differentiated behavioral regulatory effects on male and female oriental fruit flies: 16% concentrations of 3-carene, α-pinene, and β-pinene all showed significant attractant activity on both male and female adults; at the same concentrations, 4-isopropyltoluene, carvone, menthone, and eucalyptol possessed repellent effects, with 4-isopropyltoluene showing the best repellent efficacy; trans-caryophyllene and linalool showed concentration-dependent effects, with low concentrations (1%) attracting and high concentrations causing a decrease in attractant activity or even repellency; the olfactory behavior indices of male and female adults showed a highly significant positive correlation (rs=0.9802, P<0.0001), indicating a high degree of consistency in their odor recognition response patterns. Both 4-isopropyltoluene and eucalyptol exhibited dose-response activity, with 16% being the optimal concentration. Increasing the concentration to 32% and 64% did not further enhance the repellent effect, demonstrating a non-linear dose-response characteristic. Only 16% 4-isopropyltoluene and 16% eucalyptol significantly inhibited oviposition in female insects, with 4-isopropyltoluene showing the best oviposition repellency effect (OI=-0.59). Carvone and menthone showed only a minor oviposition repellency trend, while 1% trans-caryophyllene and 1% linalool significantly promoted oviposition. The olfactory behavior index of female insects was significantly positively correlated with the oviposition index (rs=0.8645, P<0.0001), indicating that the olfactory repellency level could effectively predict the oviposition inhibition effect. When 16% 4-isopropyltoluene and 16% eucalyptol were combined, the binary combination with a volume ratio of 3:1 showed better oviposition repellency activity than other ratios. However, the effects of all combined systems were weaker than those of 4-isopropyltoluene alone, clearly indicating that 4-isopropyltoluene is the core functional component of this combined system. Furthermore, 16% 4-isopropyltoluene exhibited stable and highly significant oviposition repellency effects on three typical host fruits: orange, mango, and guava, demonstrating a broad spectrum of action across hosts.

[0081] Based on existing literature, the behavioral regulation of fruit fly pests by plant-derived monoterpenoid volatiles generally exhibits compound specificity and concentration dependence. The two active substances in this invention have an optimal concentration window, eliminating the need for blindly increasing application concentrations in the field. The olfactory responses of male and female *Bacteroides citrinum* individuals are highly similar, and this repellent substance can simultaneously regulate the behavior of male and female insects in the population, demonstrating its potential for field application. There is an intrinsic neural regulatory link between olfactory perception and oviposition decision-making, allowing for rapid prediction of oviposition and repellency potential based on simple olfactory behavior screening. Existing research has confirmed that 4-isopropyltoluene possesses both repellent and oviposition-inhibiting activities against stored-product pests, while this invention demonstrates its prominent single activity against *Bacteroides citrinum*. The combination of the two substances did not produce a synergistic effect, presumably due to overlapping or competitive antagonism of their pathways. Furthermore, this substance is effective against multiple fruit tree hosts, simplifying commercial repellent formulations and reducing application costs, providing sufficient experimental evidence and theoretical support for the development of broad-spectrum plant-derived repellents for *Bacteroides citrinum*.

[0082] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A plant-derived volatile repellent for the oriental fruit fly, characterized in that, The active ingredient of the oriental fruit fly repellent is a plant-derived volatile compound, the volume fraction of which is 1%-32%, and the plant-derived volatile compound is at least one of 4-isopropyltoluene, trans-caryophyllene, carvone, menthone, eucalyptol, and linalool.

2. The oriental fruit fly repellent according to claim 1, characterized in that, The plant-derived volatiles are at least one of 4-isopropyltoluene and eucalyptol.

3. The oriental fruit fly repellent according to claim 2, characterized in that, The plant-derived volatile compound is a single 4-isopropyltoluene, and the volume fraction of the 4-isopropyltoluene is 16%.

4. The oriental fruit fly repellent according to claim 2, characterized in that, The plant-derived volatile compound is a single eucalyptol, and the volume fraction of eucalyptol is 16%.

5. The oriental fruit fly repellent according to claim 2, characterized in that, The plant-derived volatiles are a mixture of 4-isopropyltoluene and eucalyptol, with a volume ratio of (1-3):(1-3).

6. The oriental fruit fly repellent according to claim 5, characterized in that, The volume ratio of 4-isopropyltoluene to eucalyptol is 3:

1.

7. The oriental fruit fly repellent according to any one of claims 1-6, characterized in that, The repellent for the oriental fruit fly simultaneously repels both male and female adult oriental fruit flies and inhibits oviposition by females.

8. The oriental fruit fly repellent according to any one of claims 1-6, characterized in that, The oriental fruit fly repellent also includes pesticide-acceptable excipients.

9. The oriental fruit fly repellent according to claim 8, characterized in that, The excipients are selected from at least one of solvents, solubilizers, emulsifiers, stabilizers, osmotic pressure regulators, pH buffers, surfactants, diluents, and suspending agents.

10. The oriental fruit fly repellent according to claim 8, characterized in that, The oriental fruit fly repellent is any one of the following: aqueous solution, emulsifiable concentrate, microcapsule, dispersible oil suspension, granules, or ointment.

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